Investigation of Epigenetic Control of DAX-1 Expression in Human Cell Lines

Dosage-Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, Critical Region on the X chromosome, gene 1 (DAX-1 or NR0B1) is an orphan nuclear hormone receptor implicated in Adrenal Hypoplasia Congenita (AHC) and Dosage Sensitive Sex Reversal (DSS). In both instances, DAX-1 plays a key role in growth and development by modulating hormone function. In DSS, mutations on the X-chromosome lead to duplication of the region containing DAX-1, resulting in sex reversal, and in AHC, mutations in the DAX-1 gene diminish development of adrenal tissue which leads to a reduction in adrenal hormone production. Expressed predominantly in tissues such as the testes, ovaries, breast, adrenal cortex, and lung, DAX-1 may serve as an indicator of aberrant growth. Here we hypothesize that DAX-1 is epigenetically regulated, specifically in cancer cells, thereby reducing its expression. We surveyed various human cancer cells in order to determine whether inhibiting DNA methylating enzymes released epigenetic control of the DAX-1 gene, resulting in an increase in expression. By implementing molecular techniques, such as bisulfite sequencing, we determined the precise methylation sites in the DAX-1 gene. Additionally, we carried out methylation specific restriction enzyme analysis to differentiate degrees of methylation between lung, breast, liver, cervical, and adrenal carcinoma cell lines. Following confirmation of the precise methylation sites, we utilized chromatin immunoprecipitation (ChIP) in order to identify the modifying proteins present on the DAX-1 CpG islands. In conjunction with these experimental techniques, we implemented a bioinformatics approach to analyze methylation in the promoter region of the DAX-1 gene across tissue sample data acquired from The Cancer Genome Atlas Program. The results of this research could lead to a translational application of understanding where this orphan NHR fits into the development and progression of cancer. As a quickly growing field, cancer epigenetics is a key player in the ongoing pursuit for identifying biomarkers that may be pertinent in future therapeutic applications.

Identification and Analysis of a Novel NR0B1 Mutation in Late-Onset Adrenal Hypoplasia Congenita and Hypogonadism

Objective X-linked adrenal hypoplasia congenita (AHC) is a rare disorder characterized by primary adrenal insufficiency and hypogonadotropic hypogonadism (HHG) caused by mutations of the NR0B1/DAX1 gene. We aimed to search for the presence of any NR0B1/DAX1 gene mutations in a referred patient and to further characterize the phenotypes of the identified mutation. Case Presentation Herein, we report a Japanese patient with a novel missense mutation of the NR0B1/DAX1 gene resulting in adult-onset AHC and HHG. The patient was referred with diffuse skin pigmentation at 28 years of age, presented partial adrenal insufficiency and had undiagnosed incomplete HHG. Urological examination revealed severe oligospermia and testicular microlithiasis. Results The NR0B1/DAX1 gene mutation was identified by exome sequencing as a novel missense mutation (c.884A>T, p.Leu295His). We conducted in silico modeling of this mutant NR0B1/DAX1 protein (p.Leu295His) which affected the conserved hydrophobic core of the putative ligand-binding domain (LBD). In addition, functional analysis revealed that this mutant showed a decreased ability as a transcriptional repressor to suppress target genes, such as STAR and LHB. Furthermore, this mutant showed functionally impaired repression of steroidogenesis in human adrenocortical H295R cells. Conclusions We identified a novel missense mutation of the NR0B1/DAX1 gene in a patient suffering from late-onset AHC and HHG, who presented with oligospermia and testicular microlithiasis. This mutant NR0B1/DAX1 protein was found to have reduced repressor activity, according to in vitro studies, clinically consistent with the patient’s phenotypic features.

MON-076 Adrenal Hypoplasia Congenita - Is It Possible to Make This Diagnose in Previous “Idiopathic” Adrenal Insufficient Patients? Series of Cases

Background: Adrenal hypoplasia congenita (AHC) is a rare disease (1:70.000 men) characterized by reduction in all cortical adrenal hormones and also by hypogonadism. NR0B1 -related AHC includes both X-linked AHC and Xp21 deletion. AHC gene mutation was first described in 1994 and occurs in less than 1% of adrenal insufficiency (AI) cases and it is imperative that clinical endocrinologists increase their knowledge on this condition to recognize it as promptly as they were used to do so in other causes of pediatric AI.Clinical Cases: We describe here two sporadic cases and three familial cases from one family, all of them attending at our Endocrine Unit at HCPA, in the South of Brazil. The median age of AI first symptoms was 1 month old in two patients (acute infantile onset due to a salt wasting crises), and eleven years old in the remaining patients (childhood onset). Their exams confirmed the clinical suspicion of primary AI because of low plasma cortisol (from < 0.5 to 4 mcg/dl - reference range from 7-24 mcg/dl), as well as all adrenal steroids levels, high ACTH levels (from 672 to 2225 pg/ml - reference range from 7-63 pg/ml) and high plasmatic renin (from 92 to 448 mcUI/ml - reference range from 2.8-39.9 mcUI/ml). They were also extensively investigated searching for the AI pathophysiology. After some years of lost follow-up, one of the familial cases that had first been seen in 1993, returned to our hospital and was diagnosed with hypogonadism, leading to NR5OB1 gene evaluation.Regarding the genetic etiology, one of the sporadic cases developed a clinical picture including profound developmental delay, seizures and strabismus suggestive of a contiguous gene syndrome that involves Duchenne muscular dystrophy, glycerol kinase deficiency and AHC. A CGH array was performed and identified a Xp21.3-p21.1 deletion. The affected family had an extensive history suggestive of a genetic disease with X-linked inheritance pattern, as shown by the premature death of 3 uncles and a male cousin. A deleterious variant (c.131_212delinsTGAGACCTGTACCGT) in NR5OB1 gene was identified by Sanger sequencing in hemizygous state in the 46,XY affected patients.Conclusion: Albeit a rare disease, it is crucial that endocrinologist all around the World could be aware about clinical characteristics of this condition in order to properly diagnose it. And it is even possible, as it occurred with one of our patient, to make a late pathophysiologic diagnosis, making possible to treat the associated hypogonadism. Reference: Acherman JC, Vilain EJ. NR0B1- related adrenal hypoplasia congenita. GeneReviews 1993-2019.

Delayed-onset adrenal hypoplasia congenita and hypogonadotropic hypogonadism caused by a novel mutation in DAX1

In this study, we described a male who presented with delayed-onset adrenal hypoplasia congenita (AHC) and mild hypogonadotropic hypogonadism (HHG) without a relevant family history. A novel mutation in the DAX1 (dosage-sensitive sex reversal, congenital adrenal hypoplasia critical region on the X chromosome, gene 1) gene was shown to cause X-linked AHC and HHG. Genetic analysis revealed a novel nonsense mutation, c.154G > T (p.Glu52Term), in the DAX1 gene. Molecular testing demonstrated that the milder phenotype caused by this mutation was due to expression of a partially functional, amino-truncated DAX1 protein generated from an alternate in-frame translation start site (methionine at codon 83). This unusual case revealed a potential mechanism for a novel mutation that resulted in an unusual delayed-onset mild clinical phenotype. It expands the spectrum of adrenal hypoplasia congenita and hypogonadotropic hypogonadism.